Derivation and characterisation of endothelial cells from patients with chronic thromboembolic pulmonary hypertension

Derivation and characterisation of endothelial cells from patients with chronic thromboembolic pulmonary hypertension

The material obtained from pulmonary endarterectomy (PEA) offers the unique opportunity to study the pathophysiological mechanisms of chronic thromboembolic pulmonary hypertension (CTEPH) at disease site. Metabolic changes and mitochondrial disarrangements in endothelial cells might elucidate a hyperproliferative apoptosis-resistant phenotype that could explain vascular changes occurring in CTEPH. The objective of our study was to develop an in vitro model of CTEPH using patient derived cell lines and assay endothelial cell metabolism, apoptosis and potential mitochondrial disturbances.

Isolated cells obtained from freshly obstructive thromboembolic material collected at PEA (N=10), were confirmed as being endothelial cells based on cobblestone morphology, endothelial phenotype (flow cytometry, RT-PCR and immunofluorescence). Cell growth kinetics were evaluated using fold expansion, MTT assay, number of Ki67+ cells. We also measured: i) hypoxia-inducible factor 1-alpha (HIF-1a) expression by RT-PCR, ii) glycolytic enzymes and metabolites by colorimetric assays (such as lactate dehydrogenase (LDH) and lactate). iii) mitochondrial membrane potential (MMP), mitochondrial content and apoptosis/necrosis by flow cytometry and iv) mitochondrial dynamics (MD) by confocal microscopy.

Isolated cells maintained cobblestone morphology and stained positive for endothelial markers (such as CD31, VE-cadherin, eNOS and vWF (von Willebrand Factor)). They showed a hyperproliferative phenotype when compared with control human pulmonary artery endothelial cell lines (HPAE): number of Ki67⁺cells (50.33±13.4 vs 32.5±9.5; p<0.05), and fold expansion (1.56±0.08 vs 0.8±0.05; p<0.002). They also showed a reduction in the capacity to recover from a scratch in the wound-healing assay. HIF-1α was higher expressed in patient derived cells compared with HPAE (3.21±1.38 vs 1.54±0.87; p<0.001). Functionally, they showed reduced capacity to form tubule structures (150±44 vs 96±21; p<0.03). CTEPH cells showed lower rates of depolarized MMP (49.91±14.70 vs. 59.87±8.41) and a decrease of mitochondrial content (148.94±69.96 vs. 295.57±178.60).

We concluded that endothelial cells obtained from PEA in CTEPH show a hyperproliferative phenotype, metabolic disturbance, impaired function and mitochondrial material dysfunction that may play a role in the pathogenesis of pulmonary hypertension.